Long-term Objectives: The goal of this long-term project is to develop therapy against transforming growth factor-beta (TGF() to break the vicious cycle of bone metastasis. TGF( in the bone microenvironment dramatically changes the phenotype of metastatic tumor cells, causing them to make factors that stimulate pathological changes in the skeleton. We hypothesize that TGF( signaling in tumors is activated by the bone microenvironment, acts through the Smad pathway in tumor cells and can synergize with hypoxia to drive the vicious cycle to promote osteolytic bone metastases. Inhibiting both TGF( and hypoxia will provide more effective treatment of bone metastases than single agents. A secondary goal is to characterize the effect of TGF( inhibition on bone metastases due to different tumor types as well as different bone metastases phenotypes (osteolytic vs. osteoblastic). Specific Aims: Aim 1 will compare the effects of total blockade of TGF( signaling versus selective inhibition of Smad in osteolytic bone metastasis models of breast cancer, prostate cancer and melanoma and osteoblastic models of breast and prostate cancer. Aim 2 will test if bone metastases are increased by standard sex-steroid ablation treatments for breast and prostate cancer, which stimulate bone resorption and can increase TGF( in the bone microenvironment. Aim 3 will test whether hypoxia via hypoxia-indicible factor-1( (Hif1() increases TGF( signaling in tumor cells both at the molecular level in vitro and in bone metastases in vivo. Aim 4 will use new imaging procedures to demonstrate that TGF( signaling via the Smad pathway is directly and specifically activated when tumors grow in bone. Health-relatedness: Animal models of human breast cancer, prostate cancer and melanoma metastases to bone will be utilized to test drugs for effects on bone metastases that will rapidly translate to the clinic. Research Design & Methods: Three tumor types that cause both osteolytic and osteoblastic bone metastases will be studied in a mouse model. Mice will be treated with well- characterized small-molecule inhibitors of TGF(, Smads, hypoxia, and bone resorption. Tumor and bone endpoints will be evaluated by X-radiography, bioluminescent imaging, histology, and quantitative bone histomorphometry. MicroPET imaging of gene-reporter activity will provide an accurate readout of activation of TGF( signaling in tumors growing in the bone microenvironment. In vivo studies will be complemented by detailed molecular studies on actions of the TGF( and Hif effector VEGF and the TGF( inhibitory Smad7 and the corepressors Ski & SnoN and effects on TGF( -1 and Smad7 promoters. Rationale & Techniques: Animal models permit rapid preclinical testing of single agent and combination therapies to treat and prevent bone metastases and provide statistically significant endpoints of tumor growth and bone responses. Since TGF( inhibitors are now in clinical trials, it is important to understand the effects of such treatment on different tumor types and osteolytic vs. osteoblastic bone metastases. Imaging of tumor in bone permits direct validation in vivo of mechanisms that are inaccessible by other means.